Feasibility of a nerve imaging agent to reduce neurological complications of prostatectomies A key challenge in radical or nerve-sparing prostatectomies is to minimize the neurological complications that can lead to impotence in about 10%-30% of cases and incontinence in about 10% of cases. We propose to show in vivo feasibility of a novel peripheral nerve imaging agent or nerve paint comprised of a NearInfrared (NIR) dye conjugated to recombinant human Nerve Growth Factor (rhNGF) (rhNGF-NIR). The goal of this proposal is to show that rhNGF-NIR can be visualized. Our product vision is an injectable imaging agent that may be given pre-operatively to assist surgeons prior to beginning a prostatectomy. To show feasibility we selected two NIR dyes, Alexa Fluor 790 and Dyomics 831 because their chemistries (1) are similar to IRDye(R) 800CW developed by LI-COR (Lincoln, NE) whose safety is established in an FDA drug master file i.e. these agents should also be safe; (2) provide increased tissue penetration of light and an enhanced signal-to-noise background ratio relative to cyanine 5.5; (3) can be readily conjugated unlike indocyanine green (ICG); and (4) can be observed using commercial imaging systems. Rationale for NGF as targeting moiety. In preliminary studies we showed that NGF conjugated to Alexa Fluor 488 (rhNGF-488) was taken up in compartmented cultures at distal ends and moved to proximal ends. Our targeting moiety of choice is rhNGF because it is well-tolerated, non-immunogenic, and because of its dual targeting characteristics. We expect to use doses of rhNGF-NIR within the Maximum Tolerated Dose (MTD) established for rhNGF alone in clinical studies (0.1g/kg-0.3g/kg). When given sub-cutaneously (sub-c), approx. 80% of unmodified rhNGF localizes at the site of peripheral injection, is absorbed by high- affinity TrkA receptors, and moves via axonal transport to Dorsal Root Ganglia (DRG), where it degrades. Research plan. In this proposal we will reliably synthesize and characterize NGF-probes, then confirm receptor-mediated axonal transport targeted to sensory neurons (1) in vitro, in neuronal survival assays and in compartmented cultures. We will explore visualization in vivo in mouse, in real-time imaging studies. Hypothesis. We hypothesize that NGF offers a safe and selective intraneuronal targeting moiety which can deliver selected NIR dyes via peripheral injection to image peripheral nerves associated with the site of injection prior to a prostatectomy. Our Specific Aims are: Aim 1. Synthesize, optimize, and characterize dye conjugate. We will modify our proprietary linker system for rhNGF-488 to synthesize two variants (rhNGF-780, rhNGF-DYM, or rhNGF-NIR). Our goals are to (1) enable dimerization of rhNGF to optimize uptake and tissue selectivity; and (2) develop analytical protocols to yield bioactive probes with consistent protein:probe ratios. Aim 2. Bioassay for (1) neuronal survival and (2) retrograde transport in compartmented cultures. Neurons have an absolute requirement for NGF to survive. We confirm bioactivity of rhNGF-NIR variants by administering probe to mass culture of sympathetic neurons. Our first goal is to show that probes contain biologically active NGF. Next we will confirm that each rhNGF-NIR is selectively absorbed by sensory neurons. We use compartmented cultures to confirm that rhNGF-NIRs are internalized by TrkA receptors on distal ends, then transported to cell bodies in the central chamber of the culture system. Aim 3. Confirm that rhNGF-NIR can be visualized and is selectively absorbed by peripheral nerves in vivo. We will inject microdoses of rhNGF-NIR variants at the periphery and observe probes in live animals in a commercially available imaging system. In controlled studies we will inject rhNGF-NIR sub-c and im at two sites, in the footpad and in the upper haunch. Our goal here is not to model clinical application or to determine dose, but to show that rhNGF-NIRs (1) can be visualized in real-time, (2) are localized to neuronal tissue; and (3) degrade within a reasonable period after injection. If successful, we and our collaborators have the expertise to carry out IND-enabling studies distribution, dose, and toxicity studies.